Electroacupuncture stimulating Neixiyan (EX-LE5) and Dubi (ST35) alleviates osteoarthritis in rats induced by anterior cruciate ligament transaction via affecting DNA methylation regulated transcription of miR-146a and miR-140-5p

doi:10.19852/j.cnki.jtcm.2023.05.004

Abstract

Abstract:

OBJECTIVE: To explore whether electroacupuncture (EA) could alleviate osteoarthritis (OA) through affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p.
METHODS: Sixty male eight-week-old Sprague-Dawley rats were divided into three groups: normal group (normal healthy rats; no treatment), model group (OA rats; no treatment) and EA group (OA rats treated with EA). Safranin O staining and modified Mankin's score were performed to evaluate the histopathological alterations and degeneration of cartilage 8 weeks after 8 consecutive weeks of treatment. Quantitative real time polymerase chain reaction (qRT-PCR) assay was employed to evaluate the expression of miR-146a in the cartilage tissue and miR-140-5p in the synovium tissue, respectively. The bisulfite sequencing analysis and quantitative methylation specific PCR (qMSP) were used to analyze the status of methylation in the regulatory regions of miR-146a and miR-140-5p. Chromatin immunoprecipitation (ChIP) assay were performed to assess the binding of nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (SMAD-3) in the regulatory regions of miR-146a and miR-140-5p. Western blot analysis was performed to detect the expressions of DNA Methyltransferase 1 (DMNT1), DNA Methyltransferase 3A (DMNT3A), and DNA Methyltransferase 3A (DMNT3b), NF-κB, SMAD3 levels.
RESULTS: Our results showed that EA treatment significantly upregulated miR-146a and miR-140-5p expressions. qMSP analysis showed that EA significantly decreased methylation levels of miR-140-5p regulated region and miR-146a promoter in OA cartilage and synovium. Bisulfite DNA sequencing (BDS) and ChIP analysis showed that EA significantly increased binding affinity of SMAD3 and NF-kB on the hypermethylated miR-140 regulatory region and miR-146a promoter, respectively. Western Blot analysis demonstrated that EA also significantly decreased expressions of methylation related proteins- DMNT1, DMNT3a, and DMNT3b as well as NF-κB and SMAD3.
CONCLUSIONS: Electroacupuncture stimulating Neixiyan (EX-LE5) and Dubi (ST35) may alleviate OA via affecting the DNA methylation regulated transcription of miR-146a and miR-140-5p.

Key words: osteoarthritis, knee, electroacupuncture, DNA methylation, MicroRNAs

DING Luobin, WANG Huajun, LI Yao, LI Jia, LI Ling, GAO Yangping, GUAN Jian, GENG Weiqiang. Electroacupuncture stimulating Neixiyan (EX-LE5) and Dubi (ST35) alleviates osteoarthritis in rats induced by anterior cruciate ligament transaction via affecting DNA methylation regulated transcription of miR-146a and miR-140-5p[J]. Journal of Traditional Chinese Medicine, 2023, 43(5): 983-990.

Figures/Tables 2

Figure 1 EA Impacts on cartilage morphology by Safranin O staining (x 100) A: normal cartilage stained by Safranin O staining; B: OA cartilage stained by Safranin O staining Samples were collected 8 weeks after surgery C: EA treated OA cartilage. The stimulation was performed 30 min of every day for 8 weeks. OA: osteoarthritis; EA: electroacupuncture.

Figure 2 Expression of DMNT proteins and SMAD3 and NF-κB among normal, OA and EA treated group A: cartilage tissue; B: synovium tissue. Samples were collected 8 weeks after surgery. EA stimulation was performed 30 min of every day for 8 weeks DMNT: DNA Methyltransferase; SMAD3: signal transducer and activator of transcription 3; NF-κB:nuclear factor-kappa B; OA: osteoarthritis; EA: electroacupunture.

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